The long term objective of this research is to develop an interdisciplinary approach to enhance Human mesenchymal stem cell (hMSC) osteogenic differentiation in vitro, in order to advance cell-based therapies for degenerative bone diseases such as osteoporosis. Human mesenchymal stem cells (hMSCs) are precursor cells that form and heal nearly all of the mechanical tissues in humans, including bone. hMSCs are now being isolated from adults to explore whether these cells can be differentiated into osteoblasts in vitro and re-implanted as a cellular therapy to arrest or even reverse degenerative bone diseases such as osteoporosis. While some initial promising progress has been made in demonstrating the mechanoresponsive regulation of hMSC osteogenesis by matrix rigidity and external mechanical forces, in vitro cell culture conditions for hMSC osteogenesis still remain suboptimal. We hypothesize that a versatile in vitro cell culture system that allows for a rapid and reversible dynamic mechanical regulation of cell culture environment will enable an improved control of osteogenic differentiation of hMSCs. To enhance in vitro hMSC osteogenesis and further aid in mechanistic investigation of the mechanotransductive system in hMSCs, we propose to develop a novel, interdisciplinary approach combining two novel microengineering techniques developed from laboratories of the two co-PIs of this proposal, to precisely modulate dynamic subcellular mechanical forces while simultaneously measuring live- cell subcellular responses of cytoskeleton contractility of hMSCs. These novel tools include 1) A standardized library of elastomeric micropost arrays to precisely regulate substrate rigidity and as non-destructive live-cell traction force sensors for subcellular quantification of cytoskeleton contractility; and 2) A novel acoustic tweezer capable of generating controlled mechanical forces to specific adhesion receptors on cell membrane, as a unique strategy to apply external forces affecting cytoskeleton contractility. Our specific aims are: 1) To characterize spatiotemporal changes of hMSC cytoskeCSK contractility induced by ultrasound tweezers; 2) To characterize how ultrasound tweezers exert mechanical perturbations to regulate osteogenic differentiation of hMSCs; and 3) To characterize how RhoA/ROCK/myosin signaling axis is involved in intracellular force transduction from ultrasound tweezers in hMSCs. Results from this research are expected to advance our current understanding of mechanotransduction in hMSCs to provide a pivotal foundation for enhancing their osteogenic differentiation. Improved understanding the mechanotransduction system in hMSCs may provide fundamental insights into hMSC biology, as well as practical approaches to improve hMSC differentiation in vitro for cell-based therapeutic applications for treating bone diseases.